Antibodies directed against binding-associated epitopes

ABSTRACT

Binding of two members of a binding couple reveal epitopes which art revealed only after binding and antibodies directed against these epitopes bind to the bound couple at a significantly higher affinity than their binding affinity to either of the two members themselves when not bound to one another. The novel epitope and the antibodies of the invention have various therapeutic and diagnostic applications such as treatment and immunization against viral diseases.

FIELD OF THE INVENTION

[0001] The present invention concerns novel antigenic epitopes whichbecome substantially more accessible after binding of two members of abinding couple, e.g. ligand-receptor binding, antibody-antigen binding,etc. These novel antigenic epitopes will be referred to herein at timesas “binding” associated epitopes (BAE). A specific aspect of the presentinvention concerns BAE which are revealed after virus-receptorinteraction, e.g. HIV-CD4 interaction.

[0002] The present invention further concerns antibodies, particularlymonocional antibodies, directed against BAEs, and further concerns theuse of such antibodies or BAEs in diagnostics and treatment.

BACKGROUND OF THE INVENTION AND PRIOR ART

[0003] Binding of two members of a binding, couple, e.g. a virus to itsreceptor on a cell membrane, is a complex interaction which may involve,inter alia, a conformational change in the receptor and likely also inthe viral receptor-binding protein. The study of such conformationalchanges may have various important therapeutic implications.

[0004] A virus-receptor interaction which has been studied extensivelyin recent years is that of the HIV (Human Immunodeficiency Virus) to theCD4 protein which is expressed by and present on membranes of Tlymphocytes, some macrophages and likely also on several other kinds ofcells. An HIV protein, gp120, which has a binding affinity to the CD4receptor was discovered, and the receptor recognition sites in thisprotein have been at least partially identified. Seeing that the bindingbetween the HIV virus or its gp120 protein to the CD4 receptor and theoccurrences following such interaction are critical phases in theinfection process, it is believed that agents which will interfere withthese infection stages will likely be useful as drugs in treating AIDSand particularly in inhibiting the progress of the HIV infection. It hasbeen proposed to use antibodies which recognize either the CD4 receptor,the gp120 protein or the complex which is formed following binding, asit was believed that such antibodies may form useful agents ininhibiting the infection process. Monoclonal antibodies (mAbs) usefulfor this purpose have been proposed, amongst others, by Celada et al.1990 (J. Exp. Med. 172, 1143-1150), Celada. 1992 (WO 92/05799) andHealey et al. 1990 (J. Exp. Med. 172, 1223-1242) These referencesdisclosed antibodies directed against CD4 which were shown to preventsyncytium formation without interfering with the gp120/CD4 complexformation. However, all the antibodies described to date were not foundto be useful in treatment since they either bind well to CD4-receptorsand-thus may interfere with the normal function of non-infected CD4bearing cells or, where the antibodies were directed to an epitope inthe virus and specifically in the gp120protein, they were as a rulefound to be strain and even isolate-specific.

OBJECTS OF THE INVENTION

[0005] It is an object of the present invention to provide antigenicepitopes associated with binding of two members of a binding couple toone another (BAE).

[0006] It is another object of the present invention to provide bindingassociated antibodies capable of binding to a complex consisting of twomembers of a binding couple, with a higher affinity than to each memberby itself.

[0007] It is another object of the present invention to providemedicinal and diagnostic uses of such epitopes or antibodies.

[0008] The remaining objects of the present invention will be revealedin the following description and claims.

GENERAL DESCRIPTION OF THE INVENTION

[0009] The present invention is based on the surprising finding thatupon binding of two members of a binding couple, certain novel antigenicepitopes are revealed or exposed and as a result become accessible toantibodies. When a complex of the two members is injected to an animal,an immune reaction is elicited and some of the produced antibodies aresuch which bind to the complex with a substantially higher affinity thanto either of the two members individually.

[0010] Hybridomas producing such antibodies can be prepared andmonoclonal antibodies produced by such hybridomas may be used for theisolation of the epitopes and for various diagnostic and therapeuticpurposes.

[0011] The epitopes by themselves may be utilized for producing specificantibodies or in some cases for vaccination.

[0012] The novel epitopes of the invention may consist of an amino acidsequence present in one of the two members of the binding couple whichbecomes accessible to antibodies or resumes a new conformation afterinding of the two members to one another; or may consist of a pluralityof sequences either all in one member or being distributed between thetwo members but become associated with one another to form an antigenicepitope, after binding of the two members to one another.

[0013] The present invention thus provides, by one of its aspects, anantigenic epitope which is a member of a group consisting of:

[0014] (i) an epitope consisting of an amino acid sequence in a memberof a binding couple, which becomes substantially more accessible toantibodies or resumes a new conformation after binding of the twomembers to one another.

[0015] (ii) an epitope consisting of two or more amino acid sequences ina member of a binding couple which upon binding of the two members,become closely associated to form an antigenic epitope, and

[0016] (iii) an epitope consisting of two or more amino acid sequences,at least one being in one member of a binding couple, and at least oneother being in the other member of the binding couple and upon bindingof the two members, said two or more amino acid sequences become closelyassociated with one another to form an antigenic epitope;

[0017] said antigenic epitope being immunogenic.

[0018] An epitope of the kind defined under (i) will be referred toherein at times as “linear revealed epitope”; an epitope of the kinddefined under (ii) as a “discontinuous revealed epitope” and an epitopeof the kind defined under (iii) will be referred to herein at times as“combination epitope”.

[0019] The novel BAE may be an epitope which is revealed or exposed inan immunocomplexed antigen, i.e. in an antibody-antigen complex; afterligand-receptor bindings, e.g. hormone-receptor,neurotransmitter-receptor, toxin-receptor, virus-receptor bindings; etc.A specific embodiment of the present invention concerns an epitope whichis revealed after binding of a virus to its receptor, in particularepitopes which are revealed or exposed after binding of HIV through itsgp120 protein to a soluble or membrane associated CD4 receptor protein.Another embodiment concerns an immunocomplexed gp120 epitope, i.e. anepitope which is revealed or exposed after binding of gp120 to anantibody against it produced in the body during an immune reactionfollowing an HIV infection.

[0020] The present invention further provides, by another of itsaspects, antibodies which bind to a complex consisting of two members ofa binding couple with a substantially hither affinity than with each ofthe two members by themselves. A specific embodiment of this aspect ofthe invention concerns antibodies which bind to a complex formed betweenthe HIV gp120 protein and the CD4 protein and such which bind to animmunocomplexed gp120 with a substantially hither affinity than toeither of the members of the complex by themselves.

[0021] A higher affinity of binding may be 5 fold, preferably 10 foldhigher affinity of binding to the complex as compared to bindingaffinity of the antibody to each of the members of the complex bythemselves, as tested by at least one standard assay such as ELISA, RIA(Radioimmunoassay), or by means of a FACS (Fluorescent Activated CellSorter) analysis. It should be noted that at times higher affinity ofbinding may be seen by such standard procedures, but may not be seen tothe same extent in other experimental procedures. For example, a crypticBAE normally effectively revealed after complex formation may also alsobe exposed in a protein without complex formation when, for example,denatured on a SDS gel. In case the test is performed on proteins on anSDS gel, a higher affinity of binding to the complex may not be seen,although present in the non-denatured proteins.

[0022] The antibodies of the invention may be poly- or monoclonal,although for reasons of high specificity and ease of obtainingrelatively large quantities, monoclonal antibodies are generallypreferred.

[0023] The epitopes of the invention may be detected and isolated byvarious methods, some of which will be briefly detailed herein:

[0024] 1. Western blotting analysis: CD4 or gp120 are digested by anumber of proteolytic enzymes. The resulting proteolized fragments aregel electro-phoresed on SDS acrylamide gels, and blots are prepared bytransferring the separated fragments from the gel to a suitable filter.The filter is then probed with a series of labelled mAbs. A fragmentthat repeatedly binds to a mAb of interest is transfered to a PVDF (polyvenil difluoride—Millipore Inc. Ma.) membrane and is then sequenced byany of a number of sequencing methods known per se.

[0025] 2. Pepscan analysis: (see Geysen et al., 1984. Proc. Natl. Acad.Sci. U.S.A., 81: 3998-4002; Geysen et al., 1985. Proc. Natl. Acad. Sci.,U.S.A., 82: 178-182). A series of synthetic peptides corresponding tothe complete sequence of the CD4 or gp120 molecules are produced inmultiwell ELISA plates by solid phase Merrifield peptide synthesis. Thesynthetic peptides are then screened by incubating the plates withlabelled mAbs of interest. An antibody that binds to a specific peptideis therefore mapped to that corresponding sequence.

[0026] This method is suitable for linear revealed epitopes and isobviously not suitable for mapping discontinuous epitopes or combinationepitopes. In order to identify discontinuous or combination epitopes,the following method may be used.

[0027] 3. Epitope Libraries: (see Scott et. al. 1990. Science 249:386-390; Delvin et al., 1990, Science 249: 404-406; Cwirla et al., 1990,Proc. Natl. Acad. Sci. U.S.A. 87: 6378-6382). A library consisting of acollection of the entire repertoire of combinations of peptide sequencespresented on the surface of filamentous phases is constructed. (Thus,for example, the complete collection of hexapeptides is 20°=6.4×10⁷peptides). The phage containing an epitope of interest is then enrichedby a Biopanning method in which a few microliters containing the entirelibrary are first incubated with a suitable mAb in a flask. Thelibrary-mAb mixture is then transferred to a petri dish containingimmobilized streptavidin. Only phages bound by the biotinylated mAb willbind to the streptavidin in the dish and after washing away of thenon-bound phages, the bound phase is grown in the plate and ultimatelysequenced to reveal the desired epitope.

[0028] The method under 3 is particularly suitable for the detection ofdiscontinuous epitopes or combination epitopes, due to the existence, insuch libraries, of “mimetopes”, i.e. linear peptides that functionallymimic such which can naturally be produced by discontinuous distantresidues.

[0029] In order to prepare the antibodies of the present inventionlaboratory animals are injected with complexes formed between the twomembers of a binding couple such as complexes formed between viruses orviral particles and the receptor to which they bind, e.g, gp120/CD4complexes in the case of the HIV or with immunocomplexed viruses orviral particles, e.g. immunocomplexed gp120. Following injection and thedevelopment of an imune reaction, spleen cells may be isolated fromthese laboratory animals and hybridomas may then be prepared by methodsgenerally known per se. The hybridomas are then screened for such whichsecrete antibodies which react with the complex with higher affinitythan with each of the individual components.

[0030] Hybridomas producing monoclonal antibodies of the inventionconstitute another aspect of the present invention. One hybridoma cellline designated hereinbelow as CG-10 was deposited on Feb. 4, 1993, atthe European Collection of Animal Cell Culture (ECACC), Porton Down,Salisbury, Wiltshire, SP4 OJG, United Kinpdom, and was assigned theAccession No 93020415.

[0031] The antibodies of the invention can be used as a therapeuticagent for a variety of applications such as for the treatment of viralinfections in order to inhibit further progagation of the infection. Forexample, antibodies which are specific for gp120/CD4 complexes may havean important potential use in the treatment of AIDS. As known, for humanmedicinal use, the mAbs should preferably be “humanized”, e.g. bymethods known per se such as CDR loop grafting (see Verhoyen et al.,1988, Science 239: 1534-1536). Alternatively, the mAbs should be ofhuman origin, i.e. human mAbs. Additionally, antibodies of the inventionmay have various diagnostic applications, e.g. anti-immunocomplexedgp120 be used for diagnosis or staging of HIV infections.

[0032] The epitope of the invention may have various uses such as indiagnostics, as well as in immunization. For similar uses alsoanti-idiotype antibodies against the above antibodies of the inventionmay be used. Such anti-idiotype antibodies also constitute an aspect ofthe invention.

[0033] In the following description specific reference will at times bemade to HIV-related epitopes which become accessible to antibodies uponbinding of HIV or its gp120 protein to the CD4 protein and to ananti-immunocomplexed gp120 antibody and to antibodies which specificallybind to complexes formed between gp120 and CD4 proteins or bind toimmunocomplexed gp120. It will no doubt be appreciated by the artisanthat although these embodiments of the present invention are preferredembodiments, the invention is not limited thereto.

[0034] Antibodies available to date which were proposed for use in AIDStreatment were unsuitable for this purpose. On the one hand, prior artantibodies directed against different epitopes of gp120, were found tobe ineffective in inhibiting viral infections particularly in view ofthe very high strain and isolate variability of this protein. On theother hand, prior art antibodies directed against the CD4 protein mightinterfere with the normal functions of non-infected CD4-expressingcells. Against this, the antibodies of the present invention do notpossess these drawbacks associated with prior art antibodies. Theantibodies of the invention, in the specific case of HIV, arespecifically directed to epitopes which are revealed or become moreaccessible after interaction between the HIV and the CD4 protein, andwill thus inhibit progress of the infection, e.g. the formation ofsyncytia of lymphocytes, without interferring with the normal functionsof non-infected CD4-expressing cells. Furthermore, such antibodies arealso not likely to be strain or isolate specific, since even if theepitope is of a viral origin, the fact that it is not accessible priorto binding means that it is not subject to selective forces as in thecase of normally exposed epitopes. In addition, if the antibodies are ofa suitable kind, their binding to the complex may also elicit a cellularcytotoxic immune response against the infected cells.

[0035] The antibodies of the present invention may be conjugated toradioactive or cytotoxic substances. Such conjugated antibodies willlocalize only on infected cells and will thus serve as specific targetedchemotherapeutic agents and will destroy only infected cells withoutseverely damaging normal, non-infected cells of the same kind.

[0036] Furthermore, anti immunocomplexed gp120 mAbs may serve in thediagnosis of HIV infections. To enable their use in detection in adiagnostic procedure, such antibodies may preferably be conjugated tovarious markers, such as radioactive or fluorescent substances, orenzymes such as horseradish peroxidase etc.

[0037] By another aspect of the present invention, there is provided apharmaceutical composition for treating a viral infection comprising, asan active agent, an antibody according to the invention.

[0038] By another aspect of the invention there is provided an antiviralvaccine comprising as an active agent, an epitope of the invention.

[0039] By another aspect, the present invention provides a method fortreating a viral infection, comprising administering to a patient aneffective amount of an antibody according to the invention.

[0040] By another aspect, the present invention provides a method forthe diagnosis or staging of a viral infection, utilizing the antibodiesof the invention. By one embodiment the method comprises contactingcells susceptible of being infected by the virus with an antibody of theinvention. and then detecting the presence of such antibodies on thecells' surface. By another embodiment, the method comprises contacting abody fluid sample with an antibody against an immunocomplexed virus orviral particle or with such an antibody conjugated with a detectablemarker and then detecting the formation of immunocomplexes with saidantibody or conjugate. Where the antibodies of the present invention arenot conjugated to a detectable marker, a second antibody directedagainst the antibodies of the invention, conjugated to a detectablemarker will typically be introduced for the assay.

[0041] By another aspect of the invention there is provided a method forimmunizing an animal against a viral infection comprising administeringto a subject an effective amount of a BAE epitope of the invention, of akind which is revealed after binding of a virus to its receptor.

[0042] The invention will now be described with reference to specificembodiments described in the following examples, with reference at timesto the Figures in the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 shows a series of scatchard plots obtained with fivedifferent antibodies reacted with either the complex formed betweengp120 and CD4 or with either of these two agents by themselves: a)antibody CG-4; b) antibody CG-9; c) antibody CG-10; d) antibody CG-25;and e) antibody CG-76.

[0044]FIG. 2 shows the affinity of binding of four of the antibodies toeither the complex or to CD4.

[0045]FIG. 3 is a micrograph showing syncytium formation between CD4expressing CEM cells and gp120 expressing BSC1 cells without antibodies(A) or in the presence of the various monoclonal antibodies (B-I).

EXAMPLES Example 1

[0046] Production of CD4/gp120 Complexes

[0047] Recombinant gp120 produced in a baculavirus expression system wasobtained from American Bio-Technologies. Recombinant soluble CD4,produced in Chinese hamster ovary (CHO) cells, was obtained from DuPont.CD4/gp120 complexes were prepared by a number of procedures. Forexample, 100 μg of gp120 can be mixed with 50 μg of CD4 in Tris bufferedsaline (TBS) at 4° C. for 12 hours. The complex is then dialysed againstthe same buffer prior to injection.

[0048] In order to verify that a CD4/gp120 complex was formed, ELISAassays were used. In this assay, either gp120 or CD4 were immobilized ona plate and then a soluble counterpart (CD4 or gp120, respectively) wasadded to the plate. Subsequently, the plates were probed with a firstantibody being either anti-gp120 (obtained from AmericanBio-Technologies) or OKT4 and OKT4A (which are anti-CD4 antibodiesobtained from Orthodiagnostics Inc.) followed by a second alkalinephosphatase conjugated anti-mouse antibody.

[0049] For the ELISA assay Costar EIA/RIA 96 well plates (N3590) werecoated with 50 μl of various concentrations of gp120 or CD4 in TBS(tris-buffered saline) overnight at 4° C. The plates were then washed inTBS and blocked with 3% BSA in TBS for 1 hour at room temperature (RT).Then 50 μl of 5 μg/ml solution of either recombinant CD4 or recombinantgp120 was added into the wells. The wells were rinsed and theappropriate mAb in 0.3% BSA/TBS was added to them and incubated at RTfor 2-3 hours. The wells were then washed with TBS and the secondantibody (alkaline phosphatase conjugated goat anti-mouse antibody[Sigma. A-0162]) was added (1:1000 in 0.3% BSA/TBS) and incubated for 1hour at RT. After washing the wells, they were reacted withp-nitrophenyl phosphate (1 mg/ml in 1M diethanolamine buffer pH 9.8/0.5mM MgCl₂) and read at 405 nm.

[0050] Exemplary results of such a complex analysis is shown in thefollowing Table I (the numbers in the Table represent OD×10⁻³): TABLE IPlated Plated gp120 CD4 μg/ml OKT4A* OKT4 μg/ml OKT4A OKT4 10.0 27 6405.0 206 729 5.0 23 773 2.5 135 492 2.5 27 313 1.25  42 291 1.25 29 1160.60  22 138

Example 2

[0051] Production of Anti-complex mAbs

[0052] 3 mice were immunized with a CD₄/gp120 complex that had beenextensively dialyzed. A total volume of 1 ml complex prepared as inExample 1, was dialyzed against 2 liters of Tris buffered saline (TBS),(total volume 6 liters) for 12-14 hours at 4° C. These mice developed agood immune response against both CD4 and gp120.

[0053] A plurality of hybridoma cell lines were prepared from thesemice. From the spleen of one of the injected mice, which was found to beextremely large, about 4×10⁸ cells were obtained and 4 aliquots of about10⁸ cells were taken separately. Each aliquot was fused with NS-1 cells.Two fusions were processed in parallel. A total of 1170 clones wereobtained and after 10 days of culture, the media were screened forantibodies directed against CD4/gp120 complex in an ELISA essay whichwas similar to that described above in Example 1 with a difference inthat a CD4/gp120 complex was immobilized on the plates (5 μg gp120: 2.5μg CD4/ml).

[0054] 147 clones were found positive and these were then rescreenedwith immobilized CD4/gp120 complex as well as separate immobilized CD4and gp120 in a similar ELISA assay to the above. Of the original clonesonly 81 continued to secrete antibodies and of these 15 were selectedfor future characterization. Out of these, 13 clones were found to bestable and were injected into mice for a successful production ofascites fluids.

[0055] Ascites fluids of all 13 mAbs described were produced,lyophilized and analyzed for their ability to bind CD4, gp120 andCD4/gp120 complexes. The mAbs were tested by ELISA assays, FACS analysisand Western Blots. A collection of 10 mAbs was identified as beinginteresting for further analysis and the result of their binding studiesis shown in the following Table II. TABLE II mAb subclass gp120/CD4gp120 CD4 1 CG-1  IgG1 +++ − +/− 2 CG-4  IgG1 +++ +++ − 3 CG-7  IgG1 +++− +/− 4 CG-8  IgG1 +++ − +/− 5 CG-9  IgG1 +++ − ++ 6 CG-10 IgG1 +++ − −7 CG-25 IgG1 +++ − ++ 8 CG-30 IgG1 +++ − ++ 9 CG-40 IgG1 +++ +++ − 10 CG-76 IgG1 +++ − +++

[0056] Of these antibodies, several including those designated CG-1,CG-7, CG-8, CG-9, CG-10, CG-25 and CG-30, are such having thecharacteristics of the antibodies of the present invention. Of thoseantibodies, CG-10, as can be seen from the above results, is specificonly for the CD4/gp120 complex.

Example 3

[0057] Scatchard Analysis of Five mAbs.

[0058] A series of scatchard analyses were performed in order todetermine the binding affinity of five mAbs (CG-4, CG-9, CG-10, CG-25and CG-76) for the CD4/gp120 complexes as compared to their bindingaffinity for isolated CD4 or gp120.

[0059] CD4, gp120 and CD4/gp120 complexes were immobilized in wells ofELISA plates. The tested mAbs were iodinated with ¹²⁵I and theimmobilized antigens were incubated with the iodinated mabs. Thescatchard plots of the five above mentioned mAbs are seen in FIG. 1. Theaffinity of binding of four of the mAbs to either the complex or to theCD4 is shown in FIG. 2.

[0060] mAbs, CG4 and CG-76 showed similar binding affinities to isolatedCD4 and CD4/gp120 complexes and may therefore be of the type previouslyreported by Celada et al.

[0061] As opposed to this, mAbs CG-9 and CG-25 showed a respective 10and 100 fold higher binding affinity to the CD4/gp120 complex, ascompared to their binding affinity to CD4 alone. Furthermore, mAb CG-10had no affinity for the isolated CD4 and bound exclusively to theCD4/gp120 complex.

Example 4

[0062] Inhibition of Syncytium Formation

[0063] The formation of syncytia between vaccinia BSC1 cells (Africangreen monkey kidney cells) infected with the recombinant vaccinia cloneVEP16 (see Ashorn et al., 1990 J. Virol. 64 2149-2156) expressing gp120on their surface and CEM cells (a human T helper lymphocyte cell line)expressing CD4 on their surface was tested. Generally, BSC1 cultureswere infected with recombinant vaccinia (5 pfu/ml) expressing cellsurface gp120. These cells were then mixed with CEM cells in thepresence of varying amounts of mAbs and incubated for different periodsof time. The degree of syncytia formation was monitored and thus theextent of neutralization potential for the various mAbs was estimated.

[0064] As is shown in panel A of FIG. 3, when infected BSC1 cells weremixed with CEM cells, syncytia were formed within a few hours. Thepotential of the different antibodies to neutralize syncytium formationwas tested by pre-incubation of CEM cells for 5 to 12 hours with thetested mAbs before, the addition of the infected BSC1 cells (panels C,G, H and I) or by the addition of the tested mAbs simultaneously withthe BSC1 cells (panels B, D, E and F).

[0065] The results are shown for the following mAbs which were added inthe indicated amounts:

[0066] Panel B: 10 μg of mAb CG-9; Panel C: 1μ of mAb, CG-25; Panel D &G: 1 μg of CG-76; Panel H: 1μ of mAb CG-10; and Panel I: 1 μg of CG-76.

[0067] As seen in FIG. 3, all the tested mAbs (CG-9, CG-10, CG-25 andCG-76) showed at least some syncytium neutralizing activity, each to adifferent extent. In addition, the pre-incubation of the mAbs with CD4expressing CEM cells improved the neutralizing, activity of the mAbs.

Example 5

[0068] Preliminary Analysis of Sera Obtained from HIV+ HemophiliaPatients

[0069] Sera from five HIV+ hemophilia patients were tested for theirpotential to inhibit the binding of several mAbs to CD4/gp120 complexesin a competitive ELISA.

[0070] For the competitive ELISA assay Costar EIA/RIA 96 well plates(N3590) were coated with 50 μl of 5 μg/ml CD4/gp120 complex in TBS(tris-buffered saline) overnight at 4° C. The plates were then washed inTBS and blocked with 3% BSA in TBS for 1 hour at room temperature (RT).Then, the wells were rinsed and 50 μl of the appropriately diluted mAbin 0.3% BSA/TBS was added to them with 50 μl of TBS (control wells) or50 μl of the mAb and 50 μl of the tested serum were added simultaneously(test wells) and incubated at RT for 2-3 hours. The wells were thenwashed with TBS and the second antibody (alkaline phosphatase conjugatedgoat anti-mouse antibody [Sigma, A-0162]) was added (1:1000 in 0.3%BSA/TBS) and incubated for 1 hour at RT. After washing the wells, theywere reacted with p-nitrophenyl phosphate (1 mg/ml in 1M diethanolaminebuffer pH 9.8/0.5 mM MgCl₂) and read at 405 nm.

[0071] The results of the competitive ELISA are shown in Table 3. TABLEIII #mAb Pat. #1 Pat. #5 Pat. #8 Pat. #9 Pat. #11 CG-1  50 40 55 70 0CG-4  78 87 68 75 60  CG-7  59 61 68 70 0 CG-8  43 60 58 71 0 CG-9  1138 20 27 0 CG-10 65 87 98 91 68  CG-25 25 44 22 41 0 CG-30  0 40  0 21 0CG-40 16 44  0  8 0

[0072] The numbers (N) in the above table represent the No of inhibitioncalculated as follows: $N = {\left\lbrack {1 - \frac{\begin{matrix}{{{O.D.\quad {of}}\quad {binding}\quad {of}\quad {mAbx}\quad {to}\quad {{CD4}/{gp120}}}\quad} \\{{in}\quad {the}\quad {presence}\quad {of}\quad {the}\quad {tested}\quad {serum}}\end{matrix}\quad}{{O.D.\quad {of}}\quad {binding}\quad {of}\quad {mAbx}\quad {to}\quad {{CD4}/{gp120}}}} \right\rbrack \times 100}$

[0073] Thus, for example, serum from patient no. 11 inhibited 68% of thebinding of mAb CG-10 to immobilized complex.

[0074] The results in Table III show that sera from all five testedhemophilia patients can compete with the binding of mAb-CG-10 to theCD4/gp120 complex. It appears, therefore, that HIV infected hemophiliacscontain complex specific antibodies and that mAbs of the presentinvention may thus serve in the diagnosis of HIV infections.

Example 6

[0075] Detecting the Presence of gp120 in a Blood Sample using the CG-10mAb

[0076] The close proximity assay is a method for measuring the existenceof receptor/ligand binding without the need to separate bound from freeligand (Hart, H. E. and Greenwald, E. B. Mol Immunol.16, 265-267 (1979);Udenfriend, S. et al. PNAS 82, 8672-8676 (1982); Udenfriend S. et al.Anal Biochem. 161, 494-500 (1987); U.S. Pat. No. 4,568,649). This assaymakes use of beads that have been impregnated with a scintillating dyehaving a desired receptor immobilized on their surface. The beads aremixed with a radioactive ligand and while the radiation emitted bynon-bound ligand molecules in solution is quenched by the medium, theemission of a ligand which is bound by the receptor coating thescintillating beads, due to the close physical proximity, can interactwith the dye and elicit a secondary photon. This scintillation is easilymonitored and quantitated in a standard scintillation counter (in theabsence of any additional scintillating fluid).

[0077] CD4 is immobilized on the surface of commercially availablescintillating beads (Amersham product #RPN 141) using a commerciallyavailable mAb such as OKT4 or other mAbs such as CG-76 (see Example 2).

[0078] Alternatively, beads impregnated with a fluorophor (obtained fromNuclear Enterprises, Scotland, product #NE-102A) are directly coatedwith CD4 as follows: methyl groups are oxidized to COOH groups eitherwith dilute nitric acid or with KMnO₄ and these are used to couple theCD4 via the water soluble carbodiimide reaction. The beads are washed toseparate excess carbodiimide and unreacted CD4.

[0079] A tested blood sample is solubilized with Triton-X-100 andcleared from erythrocytes by methods known per se. The beads coated withCD4 are then mixed with the blood sample and a radioactive labeled CG-10mAb (or any other mAb directed against the CD4/gp120 complex) is addedto the mixture. The sample is read in a standard scintillation counter;a high reading indicating the presence of gp120 in the tested bloodsample.

[0080] Another application in which CG-10 could be used as a diagnosticcould employ such methods as Immuno-PCR in which the DNA template wouldbe conjugated to CG-10 immunoglobulin. This could be done with theglycomoiety of the antibody.

Example 7

[0081] Production of Anti gp120/V3 Loop Complex mAbs

[0082] A gp120/anti-HIV-1V3 loop complex was prepared by mixing a mAbdirected against anti-HIV-1 V3 loop_(IIIB) designated M77 (obtained fromAdvanced Bioscience Laboratories, Inc., MD USA-ABL) with recombinantHIV-1 gp120_(IIIB) (obtained from American Bio-Technologies-ABT). BALB/cmice were immunized with the prepared complex and hybridoma cell lineswere prepared from the injected mice. Media from several clones obtainedfrom these hybridomas were screened for antibodies directed against thegp120/anti V3 loop complex in an ELISA assay as described in Example 2.The clones that were found positive were retested for their affinity forthe above complex as well as for separate gp120 and M77 mAb. mAbs thatbound gp120_(IIIB) were also tested for their affinity of bindingHIV-2_(ST) (a preparation of recombinant gp120 derived from anHIV-2_(ST) isolate, purchased from Smithline Beecham, King of Prussia,Pa.). The results of the binding studies of several of the mAbs is shownin the following table 4. TABLE IV Clone Type Complex M77 120_(IIIB)120_(ST) 3F lgG1 1.204 0.035 0.193 — 4D lgG1 1.093 0.044 1.090 0.629 4GlgG2b 0.840 0.033 0.907 0.686 4H lgG1 1.063 0.083 0.379 0.564 7A lgM0.884 0.045 0.944 1.238 7F lgG2a 1.136 0.092 0.214 — 8G lgG1 1.149 0.0640.141 —

[0083] Of these antibodies, those designated 3F and 8G, are such havingthe characteristics of the antibodies of the present invention, i.e.specific for the gp120/anti V3 loop complex and bind at very lowaffinity to the N177 mAb and gp120_(IIIB).

1. An antigenic epitope which is a member of a group consisting of: (i)an epitope consisting of a sequence in a member of a binding couple,which becomes substantially more accessible to antibodies or resumes anew conformation after binding of the two members to one another, (ii)an epitope consisting of two or more sequences in a member of a bindingcouple which upon binding of the two members, become closely associatedto form an antigenic epitope, and (iii) an epitope consisting of two ormore sequences, at least one being in one member of a binding couple,and at least one other being in the other member of the binding coupleand upon binding of the two members, said two or more amino acidsequences become closely associated with one another to form anantigenic epitope; said antigenic epitope being immunogenic.
 2. Anepitope according to claim 1, being revealed after antibody-antigen orligand receptor binding.
 3. An epitope according to claim 2, beingrevealed after virus or viral particle-receptor binding.
 4. An epitopeaccording to claim 3, being revealed after HIV or gp120-CD4 binding. 5.An epitope according to claim 4 capable of binding to a monoclonalantibody produced by the CG-10 hybridoma deposited with the EuropeanCollection of Animal Cell Culture (ECACC) under the accessionNo.93020415.
 6. An epitope according to claim 5 being the anti-idiotypeof a mAb produced by the CG-10 hybridoma.
 7. An epitope according toclaim 4, consisting of a sequence in the gp120 protein.
 8. An epitopeaccording to claim 2, being revealed after binding of gp120 to ananti-gp120 antibody.
 9. An epitope according to claim 8, consisting of asequence present in the gp120 protein.
 10. An antibody having bindingspecificity to an epitope according to any one of claims 1 to
 9. 11. Anantibody according to claim 10, having a binding affinity to a complexformed between two members of a binding couple, which is at least 5 foldhigher than its binding affinity to either of the two members bythemselves.
 12. An antibody according to claim 10 having a bindingaffinity to a complex formed between two members of a binding couple,which is at least 10 fold higher than its binding affinity to either ofthe two members by themselves.
 13. An antibody according to any one ofclaims 10 to 12, directed against an epitope which is revealed afterbinding of the HIV gp120 protein to the CD4 protein.
 14. An antibodyaccording to claim 13, directed against an epitope which consists of asequence of the gp120 protein.
 15. An antibody according to any one ofclaims 10 to 14, being a monoclonal antibody.
 16. An antibody accordingto claim 15, being the CG-10 antibody.
 17. An antibody having a bindingaffinity similar to that of the antibody of claim
 16. 18. Ananti-idiotype antibody of a mAb according to any one of claims 10 to 17.19. A hybridoma capable of secreting a monoclonal antibody according toany one of claims 15 to
 17. 20. A hybridoma according to claim 19deposited with the European Collection of Animal Cell Culture (ECACC)under the accession number
 93020415. 21. An antibody according to anyone of claims 10 to 17, being conjugated to a cell cytotoxic substance.22. An antibody according to any one of claims 10 to 17, beingconjugated to a detectable marker.
 23. A pharmaceutical composition fortreating a viral infection comprising an antibody according to any oneof claims 10 to 17 or a conjugate according to claim 21 or
 22. 24. Amethod for treating a viral infection, comprising administering to apatient an effective amount of an antibody according to any one ofclaims 10 to 17 or a conjugate according to claim 21 or
 22. 25. A methodof diagnosis of a viral infection, comprising contacting the cellssusceptible of viral infection with an antibody of any one of claims 10to 17 and then detecting the presence of the antibodies on the cells'surface.
 26. A method according to claim 25, wherein the cells arewithdrawn from the patient and contacted with the antibodies in vitro.27. A method of diagnosis of a viral infection, comprising contacting abody fluid sample with an antibody according to any one of claims 10 to17 or with a conjugate according to claim 21 or 22 and detecting theformation of immunocomplexes involving said antibody or said conjugate.28. A method for the detection of the presence in a body fluid ofantibodies specific for an epitope according to any one of claims 1 to9, comprising contacting the body fluid or an antibody containingfraction thereof, with an anti-idiotype antibody according to claim 18.29. A method for immunizing an animal against a viral infectioncomprising administering to a subject an effective amount of an epitopeaccording to any one of claim 3 or 7 or an anti-idiotype antibodyaccording to claim 18.